This proposal focuses on the pathophysiology of cation and water homeostasis in sick red blood cells (SSRBC). Potassium depletion in these cells exceeds Na loading, and consequently SSRBC become dehydrated. This dehydration increases the cell's tendency to sickle and also directly results in abnormal deformability and rheologic properties, even in the oxygenated state. Two potential mechanisms of Na loading and K depletion will be explored: a) NaK Pump inhibition b) deoxy NaK exchange (reversible stimulation of K efflux and Na influx under deoxygenating conditions). Whether pump inhibition in irreversibly sickled cells (ISC) results from reduction of pump numbers or alteration of pump kinetics will be determined by measuring the number of pump sites with 3H-ouabain, determining the maximal pump turnover (ouabain-sensitive 86Rb update), and estimating the affinities of the pump for internal Na and external K in cells with internal cations altered by nystatin treatment. Three possible causes of NaK pump inhibition in ISC will be investigated: cellular dehydration, membrane interaction with deoxy Hbs, and elevated intracellular Ca. The hypothesis that deoxy NaK exchange in SSRBC is mediated by a countertransport mechanism will be tested. K efflux (net and tracer 86Rb) and Na influx (net and 22 Na) via this pathway will be characterized with regard to their dependence on intra- and extracellular cations, stoichiometry, and sensitivity to known counter-transports inhibitors. The kinetic properties of the deoxcy fluxes will be compared to those Na:Na (Na:Li) countertransport in normal and SSRBC. In addition, the physiologic role of deoxy NaK exchange will be assessed by determining its dependence on Hb-O2 saturation and its relative activity in density separated SSRBC population. Two hypothetical mechanisms for net cation loss and cellular dehydration will be explored: the NaK pump in concert with NaK deoxy exchange, and increased diffusional permeability of the SSRBC membrane to K. The ability of NaK deoxy exchange (moving one Na in for one K out) along with the NaK pump (moving three Na out for two K in) to mediate net cation loss from SSRBC without excessive Na loading will be analysed. The conditions under which diffusional movements of Na and K may be measured in RBC will be rigorously defined and the permeability of SSRBC to these ions will be compared to that of normal RBC.